The (b/a)8 TIM barrel motif is one of the most common in biology, supporting the catalysis of a host of biochemical reactions in organisms from all three super-kingdoms of life. Having demonstrated a primary role for chain topology in shaping the folding free energy surface of TIM barrels, the role of the amino acid sequence in guiding the rapid and efficient formation of the native conformation for a set of homologous TIM barrels will now be probed with a variety of biophysical tools. X-ray scattering (SAXS/WAXS) and Fvrster resonance energy transfer (FRET) techniques will assess global, regional and pair-wise specific dimensions in chemically-denatured states to probe for non-random structure that might influence the earliest stages of folding. Microchannel mixing devices interfaced to FRET, SAXS/WAXS and circular dichroism (CD) detection systems will enable a dimensional analysis of microsecond folding reactions and the assessment of global secondary structure during the early events in folding. A collaborative effort will be mounted to study the nano- to microsecond folding reactions of stable bab building blocks, excised from native TIM barrels, by temperature-jump (T-jump) fluorescence and infrared spectroscopy (IR). Isotope-edited T-jump IR studies on selectively mass-labeled carbonyls in the modules will test for concerted vs. sequential folding reactions. The relationship of the sequence to the structures of transient and stable folding intermediates will be explored by applying hydrogen exchange mass spectrometric techniques on peptides extracted from proteolytic digests of pulse-quench deuterium-labeled intermediates. Comparisons of the results from sets of orthologous and paralogous TIM barrels of low sequence identity will allow robust tests of several algorithms to predict the structures of the intermediates from the sequence and/or topology. The role of hither-to-fore unrecognized but very common side chain-main chain hydrogen bonding interactions in stabilizing ba and ab hairpins and, thereby, in establishing the register of the b and a elements will be probed by mutational analysis. Bio- informatics analysis of conserved and non-conserved ba and ab hairpin clamps in a database of TIM barrel structures will enable the development of hypotheses to explain the unexpectedly large contributions of a sub- set of clamps to structure and stability. This analysis may also enable the prediction of clamps in barrels of unknown structure and, thereby, enhance the prediction of structure from sequence. The results are expected to substantially increase the understanding of the mechanism by which TIM barrel proteins fold and, especially, the role of the amino acid sequence in directing this process. PUBLIC HEALTH RELEVANCE: The insights obtained and concepts developed should have wide application to the folding mechanisms of other motifs, thereby enhancing the understanding of a fundamental process in biology. The results may also prove to be useful for the prediction of structures from sequences and for the rational design of protocols for the recovery of misfolded or aggregated proteins in the biotechnology industry. Finally, the early misfolding reactions common to TIM barrels may also provide insights into pathogenic misfolding reactions.